Natural killer (NK) cells are lymphocytes distinct from B and T cells that have essential roles in innate immunity and placental reproduction. Blood-borne NK cells respond quickly to viral infection by secreting cytokines and killing virus-infected cells; uterine NK cells co-operate with fetal trophoblast cells to remodel maternal blood vessels that will nourish the fetus. Regulating human NK cell response and function is a variety of inhibitory and activating receptors that recognize HLA class I. The killer cell immunoglobulin-like receptors (KIR) recognize polymorphic determinants of HLA-A, B and C, and are encoded by family of 15 genes in the leukocyte receptor complex (LRC) on human chromosome 19. We have shown that KIR haplotypes exhibit high variability in gene content, which combines with allelic polymorphism to diversify human genotypes in a manner approaching the HLA complex on chromosome 6. KIR gene families appear specific to primates, are most complicated in humans, and are largely species specific. Aim 1 will define the variation and worldwide distribution of KIR3DL2, a polymorphic lineage II KIR gene that encodes inhibitory receptors specific for HLA- A. Our analysis at both the levels of sequence and human population will examine the evolution of the gene, the role of natural selection and the presence of selected sites, which will be further examined in assays of immunological function. The experimental and analytical methods will be based on ones that we applied successfully to KIR3DL1/S1, the other lineage II KIR and specific for the Bw4 determinants shared by HLA-A and B. Although sibling genes, KIR3DL2 and KIR3DL1/S1 have several distinctive properties that promise their comparison will reveal distinctly different evolutionary histories. Using a similar suite of methods, Aim 2 will examine variation in two contrasting inhibitory members of the leukocyte immunoglobulin-like receptors (LILR), encoded by an LRC gene family that abuts the KIR family. LILRB1 is an NK cell receptor having broad specificity for HLA class I, complementing those of the KIR, that is subverted by the UL18 protein of human cytomegalovirus to prevent the killing of virus-infected cells. We will test the hypothesis that polymorphism in LILRB1 has been selected for escaping interaction with UL18, and determine the influence the polymorphism has on the interaction with HLA class I. LILRB3, which appears exceptionally polymorphic but has yet to be assigned either ligand or function, will be studied to determine if it has been subject to selection and to identify functionally important sites on its surface. Because the LRC is so densely packed with genes involved in innate immunity and reproduction, Aim 3 will develop a high-throughput method for assessing polymorphism throughout the LRC. This method will be used to study the world's populations, to define LRC haplotypes and to analyze the linkage disequilibrium between alleles at the different genes. Overall, this investigation will combine functional immunology with genetics, phylogenetics, and statistical analysis to study the evolution and function of natural variation in a genetic complex focused on innate immunity and reproduction. PUBLIC HEALTH RELEVANCE: Natural killer cells fight viruses at early times of infection and also contribute to reproduction, by remodeling the mother's blood vessels that will nourish the fetus. To do this NK cells use highly variable sets of interacting proteins that are specified by dedicated regions of the human genome. This project will determine the variability of these genes in the human population and how these differences affect their function.